The objective of this proposal is to elucidate the molecular and biochemical events associated with transcriptional repression through an investigation of the Drosophila protein Groucho (Gro). Gro is a long- range corepressor that plays many essential roles in metazoan development and signaling pathways. It is recruited to target promoters via interactions with numerous DMA-binding repressers including Brinker, Hairy, and Dorsal. Gro contains conserved N-terminal glutamine-rich (Q) and C-terminal WD-repeat domains that are essential for Gro-mediated repression. Although the functions of the Q and WD repeat domains have been studied extensively, the central region (which includes the GP, CcN,and SP domains) of the protein has not been well characterized. This project seeks to elucidate the contributions of the central domains to Gro-mediated repression by generating and analyzing multiple Gro deletions lacking one or more of the these domains. The proposal has four specific aims. First, to determine which domains are required for repression, the deletion variants will be assayed for their ability to drive ectopic repression of Brinker targets when misexpressed in the wing disc. Second, to determine the roles of the domains in the targeting ofGro, immunofluoresence studies will be employed to examine the subcellular localization of each deletion variant. Third, to determine the roles of the domains in normal development, the extent to which each deletion variant is able to rescue the Gro loss-of-function phenotype will be determined. Fourth, to determine the contribution of each domain to the distance dependence of repression, the deletion variants will be assessed using a series of reporters in which the distance between the site of Gro recruitment and the transcriptional start site has been systematically varied. In addition to examining levels and patterns of target gene activity, the state of the target genes will be assessed by chromatin immunoprecipitation (ChIP) assays. Increased expression levels of Gro have been associated with specific types of cancer. Therefore, understanding factors involved in repression by Gro may provide insight into the events leading to tumor formation.